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At least 19 recordsLinked to original sources
PubMed1993

Novel zinc finger motif in the basal transcriptional machinery: three-dimensional NMR studies of the nucleic acid binding domain of transcriptional elongation factor TFIIS.

Transcriptional elongation provides a key control point in the regulation of eukaryotic gene expression. Here we describe homonuclear and 15N-heteronuclear 3D NMR studies of the nucleic acid binding domain of human transcriptional elongation factor TFIIS. This domain contains a Cys4 Zn(2+)-binding site with no homology to previously characterized Cys4, Cys6, or Cys2-His2 Zn fingers. Complete 1H and 15N NMR resonance assignment of a 50-residue TFIIS peptide-Zn2+ complex is obtained. Its solution structure, as determined by distance geometry/simulated annealing (DG/SA) calculations, exhibits a novel three-stranded antiparallel beta-sheet (designated the Zn ribbon). Analogous sequence motifs occur in a wide class of proteins involved in RNA or DNA transactions, including human basal transcriptional initiation factor TFIIE. A three-dimensional model of the TFIIE Cys4 domain is obtained by DG-based homology modeling. The role of the TFIIS Zn ribbon in the control of eukaryotic transcriptional elongation is discussed.

Amino Acid Sequence↗
PubMed1993

Structure of a new nucleic-acid-binding motif in eukaryotic transcriptional elongation factor TFIIS.

Transcriptional elongation involves dynamic interactions among RNA polymerase and single-stranded and double-stranded nucleic acids in the ternary complex. In prokaryotes its regulation provides an important mechanism of genetic control. Analogous eukaryotic mechanisms are not well understood, but may control expression of proto-oncogenes and viruses, including the human immunodeficiency virus HIV-1 (ref. 8). The highly conserved eukaryotic transcriptional elongation factor TFIIS enables RNA polymerase II (RNAPII) to read though pause or termination sites, nucleosomes and sequence-specific DNA-binding proteins. Two distinct domains of human TFIIS, which bind RNAPII and nucleic acids, regulate read-through and possibly nascent transcript cleavage. Here we describe the three-dimensional NMR structure of a Cys4 nucleic-acid-binding domain from human TFIIS. Unlike previously characterized zinc modules, which contain an alpha-helix, this structure consists of a three-stranded beta-sheet. Analogous Cys4 structural motifs may occur in other proteins involved in DNA or RNA transactions, including RNAPII itself. This new structure, designated the Zn ribbon, extends the repertoire of Zn-mediated peptide architectures and highlights the growing recognition of the beta-sheet as a motif of nucleic-acid recognition.

Amino Acid Sequence↗
PubMed2000

Structure of a conserved domain common to the transcription factors TFIIS, elongin A, and CRSP70.

TFIIS is a transcription elongation factor that consists of three domains. We have previously solved the structures of domains II and III, which stimulate arrested polymerase II elongation complexes in order to resume transcription. Domain I is conserved in evolution from yeast to human species and is homologous to the transcription factors elongin A and CRSP70. Domain I also interacts with the transcriptionally active RNA polymerase II holoenzyme and therefore, may have a function unrelated to the previously described transcription elongation activity of TFIIS. We have solved the structure of domain I of yeast TFIIS using NMR spectroscopy. Domain I is a compact four-helix bundle that is structurally independent of domains II and III of the TFIIS. Using the yeast structure as a template, we have modeled the homologous domains from elongin A and CRSP70 and identified a conserved positively charged patch on the surface of all three proteins, which may be involved in conserved functional interactions with the transcriptional machinery.

Amino Acid Sequence↗
PubMed1994

The transcription factor TFIIS zinc ribbon dipeptide Asp-Glu is critical for stimulation of elongation and RNA cleavage by RNA polymerase II.

The eukaryotic transcription factor TFIIS enhances elongation and nascent transcript cleavage activities of RNA polymerase II in a stalled elongation complex. By site-directed mutagenesis, we have demonstrated that invariant residues Asp-261 and Glu-262 of the nucleic acid-binding TFIIS Zn ribbon are critical for stimulation of both elongation and RNA cleavage activities of RNA polymerase II. Substitution of either of these residues inactivates both TFIIS functions, suggesting a related role in both activities. These acidic residues may participate in phosphoryl transfer reactions by a two-metal-ion mechanism in a manner analogous to Klenow fragment. The RNA polymerase II itself may contain a Zn ribbon, in as much as the polymerase's 15-kDa subunit contains a sequence that aligns well with the TFIIS Zn ribbon sequence, including a similarly placed pair of acidic residues.

Amino Acid Sequence↗
PubMed1998

A novel nuclear import pathway for the transcription factor TFIIS.

We have identified a novel pathway for protein import into the nucleus. We have shown that the previously identified but uncharacterized yeast protein Nmd5p functions as a karyopherin. It was therefore designated Kap119p (karyopherin with Mr of 119 kD). We localized Kap119p to both the nucleus and the cytoplasm. We identified the transcription elongation factor TFIIS as its major cognate import substrate. The cytoplasmic Kap119p exists as an approximately stoichiometric complex with TFIIS. RanGTP, not RanGDP, dissociated the isolated Kap119p/TFIIS complex and bound to Kap119p. Kap119p also bound directly to a number of peptide repeat containing nucleoporins in overlay assays. In wild-type cells, TFIIS was primarily localized to the nucleus. In a strain where KAP119 has been deleted, TFIIS was mislocalized to the cytoplasm indicating that TFIIS is imported into the nucleus by Kap119p. The transport of various substrates that use other karyopherin-mediated import or export pathways was not affected in a kap119Delta strain. Hence Kap119p is a novel karyopherin that is responsible for the import of the transcription elongation factor TFIIS.

Cell Nucleus↗
PubMed2004

The Etched1 gene of Zea mays (L.) encodes a zinc ribbon protein that belongs to the transcriptionally active chromosome (TAC) of plastids and is similar to the transcription factor TFIIS.

Etched1 (et1) is a pleiotropic, recessive mutation of maize that causes fissured and cracked mature kernels and virescent seedlings. Microscopic examinations of the et1 phenotype revealed an aberrant plastid development in mutant kernels and mutant leaves. Here, we report on the cloning of the et1 gene by transposon tagging, the localization of the gene product in chloroplasts, and its putative function in the plastid transcriptional apparatus. Several alleles of Mutator (Mu)-induced et1 mutants, the et1-reference (et1-R) mutant, and Et1 wild-type were cloned and analyzed at the molecular level. Northern analyses with wild-type plants revealed that Et1 transcripts are present in kernels, leaves, and other types of tissue, and no Et1 expression could be detected in the et1 mutants analyzed. The ET1 protein is imported by chloroplasts and has been immunologically detected in transcriptionally active chromosome (TAC) fractions derived from chloroplasts. Accordingly, the relative transcriptional activity of TAC fractions was significantly reduced in chloroplasts of et1-R plants. ET1 is the first zinc ribbon (ZR) protein shown to be targeted to plastids. With regard to its localization and its striking structural similarity to the eukaryotic transcription elongation factor TFIIS, it is feasible that ET1 functions in plastid transcription elongation by reactivation of arrested RNA polymerases.

Amino Acid Sequence↗
PubMed1994

Characterization of the gene encoding the human transcriptional elongation factor TFIIS.

The transcriptional elongation factor TFIIS causes stimulation of RNA polymerase II elongation and readthrough of some of the elongation blocks. We present cloning and sequence characterization of the human TFIIS gene and a pseudogene. The intron-less organization of both of these genes indicates that previously identified cDNAs which suggested the presence of an intron were the products of cloning artifacts. The gene is organized in an uninterrupted ORF which codes for 301 amino acids, whereas the pseudogene lacks an ORF able to code for a full-length protein. The potential promoter for the gene has two putative GC-box-type consensus sequences, two CCAAT-box consensus sequences, and is bounded by a human Alu sequence. Two potential transcriptional termination signal sequences downstream from the consensus polyadenylation signal are proposed.

Base Sequence↗
PubMed2005

Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity.

Arteries and veins are anatomically, functionally and molecularly distinct. The current model of arterial-venous identity proposes that binding of vascular endothelial growth factor to its heterodimeric receptor--Flk1 and neuropilin 1 (NP-1; also called Nrp1)--activates the Notch signalling pathway in the endothelium, causing induction of ephrin B2 expression and suppression of ephrin receptor B4 expression to establish arterial identity. Little is known about vein identity except that it involves ephrin receptor B4 expression, because Notch signalling is not activated in veins; an unresolved question is how vein identity is regulated. Here, we show that COUP-TFII (also known as Nr2f2), a member of the orphan nuclear receptor superfamily, is specifically expressed in venous but not arterial endothelium. Ablation of COUP-TFII in endothelial cells enables veins to acquire arterial characteristics, including the expression of arterial markers NP-1 and Notch signalling molecules, and the generation of haematopoietic cell clusters. Furthermore, ectopic expression of COUP-TFII in endothelial cells results in the fusion of veins and arteries in transgenic mouse embryos. Thus, COUP-TFII has a critical role in repressing Notch signalling to maintain vein identity, which suggests that vein identity is under genetic control and is not derived by a default pathway.

Animals↗
PubMed2006

Mechanistic studies of the mRNA transcription cycle.

We have now completed an atomic crystallographic model of the 12-subunit yeast RNA polymerase II in elongation mode, with DNA and RNA in the active-centre cleft, and the NTP substrate at the growing end of the RNA. From these studies has emerged a detailed three-dimensional view of mRNA elongation. We have extended this structural analysis to a polymerase elongation complex bound by the transcript cleavage factor TFIIS (transcription factor IIS), which is required for polymerase escape from DNA arrest sites. A detailed model of this complex reveals a single tuneable active site for RNA polymerization and cleavage, and changes in the position of the RNA and polymerase domains, reflecting the dynamic nature of the elongation complex. An additional structure of a polymerase CTD (C-terminal domain) phosphopeptide bound by the 3'-RNA processing factor Pcf11 provides insights into the coupling of transcription elongation to mRNA processing. The structure of the CTD phosphatase Scp1 trapped in an intermediary enzymatic state explains CTD dephosphorylation during recycling of the polymerase. We also recently reported the first crystal structure of a Mediator subcomplex, which reveals an extended helical fold with a conserved hinge.

Crystallography, X-Ray↗
PubMed2005

Vascular endothelial growth factor receptor-2: counter-regulation by the transcription factors, TFII-I and TFII-IRD1.

The vascular endothelial growth factor receptor-2 (VEGFR-2/KDR/flk-1) functions as the primary mediator of vascular endothelial growth factor activation in endothelial cells. Regulation of VEGFR-2 expression appears critical in mitogenesis, differentiation, and angiogenesis. Transcriptional regulation of the VEGFR-2 is complex and may involve multiple putative upstream regulatory elements including E boxes. Transcript initiation is dependent on an initiator (Inr) element flanking the transcriptional start site. The transcription factor, TFII-I, enhances VEGFR-2 transcription in an Inr-dependent fashion. TFII-I is unusual both structurally and functionally. The TFII-I transcription factor family members contain multiple putative DNA binding domains. Functionally, TFII-I acts at both the basal, Inr element as well as at several distinct upstream regulatory sites. It has been postulated that the structure of TFII-I might allow simultaneous interaction with both basal and regulatory sites in a given promoter. As TFII-I is known to act at regulatory sites including E boxes as well as at the basal Inr element, we evaluated the possibility of Inr-independent TFII-I activation of the VEGFR-2 promoter. We found that an Inr-mutated VEGFR-2 reporter construct retains TFII-I-stimulated activity. We demonstrated that TFII-I binds to both the Inr and to three regulatory E boxes in the human VEGFR-2 promoter. In addition, reduction in TFII-I expression by siRNA results in decreased VEGFR-2 expression. We also describe counter-regulation of the VEGFR-2 promoter by TFII-IRD1. We found that TFII-I is capable of acting at both basal and regulatory sites in one promoter and that the human VEGFR-2 promoter is functionally counter-regulated by TFII-I and TFII-IRD1.

Animals↗
PubMed1995

Alanine-scanning mutagenesis of human transcript elongation factor TFIIS.

TFIIS is a transcription elongation factor that binds to RNA polymerase II and allows it to transcribe through a variety of transcriptional blockages by inducing cleavage near the 3' end of the nascent transcript. Although this cleavage reaction plays a key role in the process of reactivation of transcription by TFIIS, the exact mechanism by which TFIIS promotes readthrough by RNA polymerase II is not completely understood. We therefore undertook a systematic mutagenesis of the C-terminal half of TFIIS (delta TFIIS) to evaluate the contribution of charged residues in this region to induce transcript cleavage and promote readthrough in vitro. Twenty-two delta TFIIS alanine-scanning mutants were constructed by substitution of alanine for each amino acid in clusters of charged residues in the C-terminal half of HeLa TFIIS. The ability to induce transcript cleavage and readthrough of these mutants was tested in vitro using RNA polymerase II ternary elongation complexes arrested at a block to elongation. This alanine-scanning mutagenesis analysis allowed the identification of regions or residues important for the activity of TFIIS. Many of the mutants were reduced alike in both cleavage and readthrough activities. However, in several cases there was no simple correlation between these activity reductions.

Alanine↗
PubMed1991

Stimulation of transcript elongation requires both the zinc finger and RNA polymerase II binding domains of human TFIIS.

The eukaryotic transcriptional factor TFIIS enhances transcript elongation by RNA polymerase II. Here we describe two functional domains in the 280 amino acid human TFIIS protein: residues within positions 100-230 are required for binding to polymerase, and residues 230-280, which form a zinc finger, are required in conjunction with the polymerase binding region for transcriptional stimulation. Interestingly, a mutant TFIIS with only the polymerase binding domain actually inhibits transcription, whereas a mutant in which the polymerase binding and zinc finger domains are separated by an octapeptide is only weakly active. The zinc finger itself has no effect on transcription, but in contrast to the wild-type protein, it binds to oligonucleotides. These findings suggest that TFIIS may interact with RNA polymerase II such that the normally masked zinc finger can specifically contact nucleotides in the transcription elongation zone at a position juxtaposed to the polymerization site.

Amino Acid Sequence↗
PubMed2005

A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS.

Formation of productive transcription complexes after promoter escape by RNA polymerase II is a major event in eukaryotic gene regulation. Both negative and positive factors control this step. The principal negative elongation factor (NELF) contains four polypeptides and requires for activity the two-polypeptide 5,6-dichloro-1-beta-D-ribobenzimidazole-sensitivity inducing factor (DSIF). DSIF/NELF inhibits early transcript elongation until it is counteracted by the positive elongation factor P-TEFb. We report a previously undescribed activity of DSIF/NELF, namely inhibition of the transcript cleavage factor TFIIS. These two activities of DSIF/NELF appear to be mechanistically distinct. Inhibition of nucleotide addition requires > or = 18 nt of nascent RNA, whereas inhibition of TFIIS occurs at all transcript lengths. Because TFIIS promotes escape from promoter-proximal pauses by stimulating cleavage of back-tracked nascent RNA, TFIIS inhibition may help DSIF/NELF negatively regulate productive transcription.

Animals↗
PubMed2006

Transcription factor TFII-I conducts a cytoplasmic orchestra.

In response to extracellular ligands, surface receptor tyrosine kinases and G-protein-coupled receptors activate isoforms of phospholipase C (PLC) and initiate calcium signaling. PLC can activate expression of surface transient receptor potential channels (TRPC) such as TRPC3, which modulate calcium entry through the plasma membrane. A recent paper shows that competitive binding of cytoplasmic TFII-I, a transcription factor, to PLC-gamma results in inhibition of TRPC3-mediated agonist-induced Ca(2+) entry. These results establish a novel cytoplasmic function for TFII-I.

Animals↗
PubMed1998

Yeast transcript elongation factor (TFIIS), structure and function. II: RNA polymerase binding, transcript cleavage, and read-through.

The transcriptionally active fragment of the yeast RNA polymerase II transcription elongation factor, TFIIS, comprises a three-helix bundle and a zinc ribbon motif joined by a linker region. We have probed the function of this fragment of TFIIS using structure-guided mutagenesis. The helix bundle domain binds RNA polymerase II with the same affinity as does the full-length TFIIS, and this interaction is mediated by a basic patch on the outer face of the third helix. TFIIS mutants that were unable to bind RNA polymerase II were inactive for transcription activity, confirming the central role of polymerase binding in the TFIIS mechanism of action. The linker and zinc ribbon regions play roles in promoting cleavage of the nascent transcript and read-through past the block to elongation. Mutation of three aromatic residues in the zinc ribbon domain (Phe269, Phe296, and Phe308) impaired both transcript cleavage and read-through. Mutations introduced in the linker region between residues 240 and 245 and between 250 and 255 also severely impaired both transcript cleavage and read-through activities. Our analysis suggests that the linker region of TFIIS probably adopts a critical structure in the context of the elongation complex.

Models, Molecular↗
PubMed1998

Yeast transcript elongation factor (TFIIS), structure and function. I: NMR structural analysis of the minimal transcriptionally active region.

TFIIS is a general transcription elongation factor that helps arrested RNA polymerase II elongation complexes resume transcription. We have previously shown that yeast TFIIS (yTFIIS) comprises three structural domains (I-III). The three-dimensional structures of domain II and part of domain III have been previously reported, but neither domain can autonomously stimulate transcription elongation. Here we report the NMR structural analysis of residues 131-309 of yTFIIS which retains full activity and contains all of domains II and III. We confirm that the structure of domain II in the context of fully active yTFIIS is the same as that determined previously for a shorter construct. We have determined the structure of the C-terminal zinc ribbon domain of active yTFIIS and shown that it is similar to that reported for a shorter construct of human TFIIS. The region linking domain II with the zinc ribbon of domain III appears to be conformationally flexible and does not adopt a single defined tertiary structure. NMR analysis of inactive mutants of yTFIIS support a role for the linker region in interactions with the transcription elongation complex.

Magnetic Resonance Spectroscopy↗
PubMed2005

Positive and negative regulation of the transforming growth factor beta/activin target gene goosecoid by the TFII-I family of transcription factors.

Goosecoid (Gsc) is a homeodomain-containing transcription factor present in a wide variety of vertebrate species and known to regulate formation and patterning of embryos. Here we show that in embryonic carcinoma P19 cells, the transcription factor TFII-I forms a complex with Smad2 upon transforming growth factor beta (TGFbeta)/activin stimulation, is recruited to the distal element (DE) of the Gsc promoter, and activates Gsc transcription. Downregulation of endogenous TFII-I by small inhibitory RNA in P19 cells abolishes the TGFbeta-mediated induction of Gsc. Similarly, Xenopus embryos with endogenous TFII-I expression downregulated by injection of TFII-I-specific antisense oligonucleotides exhibit decreased Gsc expression. Unlike TFII-I, the related factor BEN (binding factor for early enhancer) is constitutively recruited to the distal element in the absence of TGFbeta/activin signaling and is replaced by the TFII-I/Smad2 complex upon TGFbeta/activin stimulation. Overexpression of BEN in P19 cells represses the TGFbeta-mediated transcriptional activation of Gsc. These results suggest a model in which TFII-I family proteins have opposing effects in the regulation of the Gsc gene in response to a TGFbeta/activin signal.

Activins↗
PubMed2000

The Rpb6 subunit of fission yeast RNA polymerase II is a contact target of the transcription elongation factor TFIIS.

The Rpb6 subunit of RNA polymerase II is one of the five subunits common to three forms of eukaryotic RNA polymerase. Deletion and truncation analyses of the rpb6 gene in the fission yeast Schizosaccharomyces pombe indicated that Rpb6, consisting of 142 amino acid residues, is an essential protein for cell viability, and the essential region is located in the C-terminal half between residues 61 and 139. After random mutagenesis, a total of 14 temperature-sensitive mutants were isolated, each carrying a single (or double in three cases and triple in one) mutation. Four mutants each carrying a single mutation in the essential region were sensitive to 6-azauracil (6AU), which inhibits transcription elongation by depleting the intracellular pool of GTP and UTP. Both 6AU sensitivity and temperature-sensitive phenotypes of these rpb6 mutants were suppressed by overexpression of TFIIS, a transcription elongation factor. In agreement with the genetic studies, the mutant RNA polymerases containing the mutant Rpb6 subunits showed reduced affinity for TFIIS, as measured by a pull-down assay of TFIIS-RNA polymerase II complexes using a fusion form of TFIIS with glutathione S-transferase. Moreover, the direct interaction between TFIIS and RNA polymerase II was competed by the addition of Rpb6. Taken together, the results lead us to propose that Rpb6 plays a role in the interaction between RNA polymerase II and the transcription elongation factor TFIIS.

Amino Acid Sequence↗